Cancer Detection
As the second leading cause of death in this country, almost 600,000 people will die from cancer per year making cancer one of the most alarming of all medical diagnosis. Lifetime risks for developing invasive cancers in men and women are 50 percent and 33 percent, respectively. Expectations are that more than 1.2 million new cases of cancer will be diagnosed in the United States in 1995. Healthcare expenses for cancer in 1994 were approximately $104 billion. However, the full impact of cancer on families and society is not measured only by the amount of money spent on its diagnosis and treatment. A significant number of people are stricken with cancer in their most productive years. Cancers accounted for 18 percent of premature deaths in 1985 and in 1991 more than 9,200 women in the U.S. died from breast cancer before the age of 55.
Currently, diagnosis of cancer is based on histological evaluation of tumor tissue by a pathologist. After a cancer is diagnosed, treatment is determined primarily by the extent or stage of the tumor. Tumor stage is defined by clinical, radiological, and laboratory methods. Standardized classification systems for the staging of tumors have been developed to clearly convey clinical information about cancer patients. Staging provides important prognostic information and forms the basis of clinical studies which allow the testing of new treatment strategies. A staging system was developed (TNM staging system), which classifies tumors according to the size of the primary tumor, the number of regional lymph nodes in which cancer is found, and the presence or absence of metastases to other parts of the body. Smaller cancers with no affected lymph nodes and no distant metastases are considered early stage cancers, which are often amenable to cure through surgical resection. A common measure of prognosis is the 5-year survival rate, the proportion of patients alive five years after the diagnosis of a cancer at a given stage. While 5-year survival rates for many cancers have improved over the last few decades, the fact that some early stage cancers recur within five years or later has led researchers to explore other additional prognostic markers including histological grade, cytometry results, hormone receptor status, and many other tumor markers. Most recently, investigators have explored the use of molecular alterations in cancers as prognostic indicators.
Genetic alterations found in cancers, such as point mutations and small deletions can act as markers of malignant cells.
Detection of Minority Nucleic Acid Sequences
A number of procedures have been disclosed to detect cancer using PCR. Sidransky, et al., “Identification of ras Oncogene Mutations in the Stool of Patients with Curable Colorectal Tumors,” Science 256: 102–05 (1992) detects colon cancer by identification of K-ras mutations. This involves a PCR amplification of total DNA, cloning into a phage vector, plating out the phage, repeated probing with individual oligonucleotides specific to several different K-ras mutations, and counting the percentage of positive plaques on a given plate. This is a technically difficult procedure which takes three days to complete, whereby the ratio of mutant to wild-type DNA in the stool sample is determined. Brennan, et al., “Molecular Assessment of Histopathological Staging in Squamous-Cell Carcinoma of the Head and Neck,” N. Engl. J. Med. 332(7): 429–35 (1995), finds p53 mutations by sequencing. This specific mutation is then probed for in margin tissue using PCR amplification of total DNA, cloning into a phage vector, plating out the phage, probing with an individual oligonucleotide specific to the mutation found by sequencing, and counting the percentage of positive plaques on a given plate. Berthelemy, et al., “Brief Communications—Identification of K-ras Mutations in Pancreatic Juice in the Early Diagnosis of Pancreatic Cancer,” Ann. Int. Med. 123(3): 188–91 (1995) uses a PCR/restriction enzyme process to detect K-ras mutations in pancreatic secretions. This technique is deficient, however, in that mutations are not quantified. Similarly, Tada, et al., “Detection of ras Gene Mutations in Pancreatic Juice and Peripheral Blood of Patients with Pancreatic Adenocarcinoma,” Cancer Res. 53: 2472–74 (1993) and Tada, et al., “Clinical Application of ras Gene Mutation for Diagnosis of Pancreatic Adenocarcinoma,” Gastroent. 100: 233–38 (1991) subject such samples to allele-specific PCR to detect pancreatic cancer. This has the disadvantages of providing false positives due to polymerase extension off normal template, requiring electrophoretic separation of products to distinguish from primer dimers, being unable to multiplex closely-clustered sites due to interference of overlapping primers, being unable to detect single base or small insertions and deletions in small repeat sequences, and not being practically suitable for quantification of mutant DNA in a high background of normal DNA. Hayashi, et al., “Genetic Detection Identifies Occult Lymph Node Metastases Undetectable by the Histopathological Method,” Cancer Res. 54: 3853–56 (1994) uses an allele-specific PCR technique to find K-ras or p53 mutations to identify occult lymph node metastases in colon cancers. A sensitivity of one tumor cell in one thousand of normal cells is claimed; however, obtaining quantitative values requires laborious cloning, plating, and probing procedures. In Mitsudomi, et al., “Mutations of ras Genes Distinguish a Subset of Non-small-cell Lung Cancer Cell Lines from Small-cell Lung Cancer Cell Lines,” Oncogene 6: 1353–62 (1991), human lung cancer cell lines are screened for point mutations of the K-, H-, and N-ras genes using restriction fragment length polymorphisms created through mismatched primers during PCR amplification of genomic DNA. The disadvantages of such primer-mediated RFLP include the requirement of electrophoretic separation to distinguish mutant from normal DNA, limited applicability to sites that may be converted into a restriction site, the requirement for additional analysis to determine the nature of the mutation, and the difficulty in quantifying mutant DNA in a high background of normal DNA. Further, these procedures tend to be laborious and inaccurate.
Coupled PCR/ligation processes have been used for detection of minority nucleotide sequences in the presence of majority nucleotide sequences. A PCR/LDR process is used in Frenkel, “Specific, Sensitive, and Rapid Assay for Human Immunodeficiency Virus Type 1 pol Mutations Associated with Resistance to Zidovudine and Didanosine,” J. Clin. Microbiol. 33(2): 342–47 (1995) to detect HIV mutants. This assay, however, cannot be used for multiplex detection. See also Abravaya, et al., “Detection of Point Mutations With a Modified Ligase Chain (Gap-LCR),” Nucl. Acids Res. 23(4): 675–82 (1995) and Balles, et al., “Facilitated Isolation of Rare Recombinants by Ligase Chain Reaction: Selection for Intragenic Crossover Events in the Drosophila optomotor-blind Gene,” Molec. Gen. Genet. 245: 734–40 (1994).
Colorectal lesions have been detected by a process involving PCR amplification followed by an oligonucleotide ligation assay. See Jen, et al., “Molecular Determinants of Dysplasia in Colorectal Lesions,” Cancer Res. 54: 5523–26 (1994) and Redston, et al., “Common Occurrence of APC and K-ras Gene Mutations in the Spectrum of Colitis-Associated Neoplasias,” Gastroenter. 108: 383–92 (1995). This process was developed as an advance over Powell, et al., “Molecular Diagnosis of Familial Adenomatous Polyposis,” N. Engl. J. Med. 329(27): 1982–87 (1993). These techniques tend to be limited and difficult to carry out.
Other procedures have been developed to detect minority nucleotide sequences. Lu, et al., “Quantitative Aspects of the Mutant Analysis by PCR and Restriction Enzyme Cleavage (MAPREC)” PCR Methods and Appl. 3: 176–80 (1993) detects virus revertants by PCR and restriction enzyme cleavage. The disadvantages of MAPREC include the requirement for electrophoretic separation to distinguish mutant from normal DNA, limited applicability to sites that may be converted into a restriction site, the requirement for additional analysis to determine the nature of the mutation, and difficulty in quantifying mutant DNA in a high background of normal DNA. In Kuppuswamy, et al., “Single Nucleotide Primer Extension to Detect Genetic Diseases: Experimental Application to Hemophilia G (Factor IX) and Cystic Fibrosis Genes,” Proc. Natl. Acad. Sci. USA 88: 1143–47 (1991), a PCR process is carried out using two reaction mixtures for each fragment to be amplified with one mixture containing a primer and a labeled nucleotide corresponding to the normal coding sequence, while the other mixture contains a primer and a labeled nucleotide corresponding to the mutant sequence. The disadvantages of such mini sequencing (i.e. SNuPe) are that the mutations must be known, it is not possible to multiplex closely clustered sites due to interference of overlapping primers, it is not possible to detect single base or small insertions and deletions in small repeat sequences, and four separate reactions are required. A mutagenically separated PCR process is disclosed in Rust, et al., “Mutagenically Separated PCR (MS-PCR): a Highly Specific One Step Procedure for easy Mutation Detection” Nucl. Acids Res. 21(16): 3623–29 (1993) to distinguish normal and mutant alleles, using different length allele-specific primers. The disadvantages of MS-PCR include possibly providing false positives due to polymerase extension off normal template, requiring electrophoretic separation of products to distinguish from primer dimers, the inability to multiplex closely-clustered sites due to interference of overlapping primers, the inability to detect single base or small insertions and deletions in small repeat sequences, and not being ideally suited for quantification of mutant DNA in high background of normal DNA. In Suzuki, et al., “Detection of ras Gene Mutations in Human Lung Cancers by Single-Strand Conformation Polymorphism Analysis of Polymerase Chain Reaction Products,” Oncogene 5: 1037–43 (1990), mutations are detected in a process having a PCR phase followed by a phase involving single strand conformation polymorphism (“SSCP”) of the amplified DNA fragments. The disadvantages of SSCP include the requirement for electrophoretic separation to distinguish mutant conformer from normal conformer, the failure to detect 30% of possible mutations, the requirement for additional analysis to determine the nature of the mutation, and the inability to distinguish mutant from silent polymorphisms.
Nucleotide Conversion Fidelity
Many of the approaches to detecting the presence of a given sequence or sequences in a polynucleotide sample involve amplification of the minority sequence(s) by polymerase chain reaction (PCR). U.S. Pat. No. 4,683,202 to Mullis, et al., and R. K. Saiki, et al., Science 230:1350 (1985). In this method, primers complementary to opposite end portions of the selected sequence(s) are used to promote, in conjunction with thermal cycling, successive rounds of primer-initiated replication. The amplified sequence(s) may be readily identified by a variety of techniques. This approach is particularly useful for detecting the presence of low-copy sequences in a polynucleotide-containing sample, e.g., for detecting pathogen sequences in a body-fluid sample. However, a nonselective PCR strategy will amplify both mutant and wild-type alleles with approximately equal efficiency, resulting in low abundance mutant alleles comprising only a small fraction of the final product. If the mutant sequence comprises less than 25% of the amplified product, it is unlikely that DNA sequencing will be able to detect the presence of such an allele. Although it is possible to accurately quantify low abundance mutations by first separating the PCR products by cloning and subsequently probing the clones with allele-specific oligonucleotides (ASOs) (Saiki et al., “Analysis of Enzymatically Amplified Beta-Globin and HLA-DQ Alpha DNA with Allele-Specific Oligonucleotide Probes,” Nature, 324(6093):163–6 (1986); Sidransky et al., “Identification of Ras Oncogene Mutations in the Stool of Patients with Curable Colorectal Tumors,” Science, 256:102–5 (1992); and Brennan et al., “Molecular Assessment of Histopathological Staging in Squamous-Cell Carcinoma of the Head And Neck,” N. Engl. J. Med., 332(7):429–35 (1995)), this approach is time consuming. In contrast, allele-specific PCR methods can rapidly and preferentially amplify mutant alleles. For example, multiple mismatch primers have been used to detect H-ras mutations at a sensitivity of 1 mutant in 105 wild-type alleles (Cha et al., “Mismatch Amplification Mutation Assay (MAMA): Application to the C-H-Ras Gene,” PCR Methods Appl., 2(1):14–20 (1992)) and claims as high as 1 mutant in 106 wild-type alleles have been reported (Haliassos et al., “Detection of Minority Point Mutations by Modified PCR Technique: A New Approach for a Sensitive Diagnosis of Tumor-Progression Markers,” Nucleic Acids Res., 17:8093–9 (1989); and Chen et al., “A Nonradioactive, Allele-Specific Polymerase Chain Reaction for Reproducible Detection of Rare Mutations in Large Amounts of Genomic DNA: Application to Human K-Ras,” Anal. Biochem., 244:191–4 (1997)). However, careful evaluation suggests these successes are limited to allele-specific primers discriminating through 3′ purine-purine mismatches. For the more common transition mutations, the discriminating mismatch on the 3′ primer end (i.e., G:T or C:A mismatch) will be removed in a small fraction of products by polymerase error during extension from the opposite primer on wild-type DNA. Thereafter, these error products are efficiently amplified and generate false-positive signal. One strategy to eliminate this polymerase error problem is to deplete wild-type DNA early in PCR.
Several investigators have explored selective removal of wild-type DNA by restriction endonuclease (RE) digestion in order to enrich for low abundance mutant sequences. These RFLP methods detect approximately 1 mutant in 106 wild-type or better by combining the sensitivity of polymerase with the specificity of restriction endonucleases. One approach has used digestion of genomic DNA followed by PCR amplification of the uncut fragments (RFLP-PCR) to detect very low-level mutations within restriction sites in the H-ras and p53 genes (Sandy et al., “Genotypic Analysis of Mutations in Taq I Restriction Recognition Sites by Restriction Fragment Length Polymorphism/Polymerase Chain Reaction,” Proc. Natl. Acad. Sci. USA, 89:890–4 (1992); and Pourzand et al., “Genotypic Mutation Analysis by RFLP/PCR,” Mutat. Res., 88(1):113–21 (1993)). Similar results have been obtained by digestion following PCR and subsequent amplification of the uncleaved DNA now enriched for mutant alleles (PCR-RFLP) (Kumar et al., “Oncogene Detection at the Single Cell Level,” Oncogene 3(6):647–51 (1988); Kumar et al., “Designed Diagnostic Restriction Fragment Length Polymorphisms for The Detection of Point Mutations in Ras Oncogenes,” Oncogene Res. 4(3):235–41 (1989); and Jacobson et al., “A Highly Sensitive Assay for Mutant Ras Genes and its Application to the Study of Presentation and Relapse Genotypes in Acute Leukemia,” Oncogene, 9(2):553–63 (1994)). Although sensitive and rapid, RFLP detection methods are limited by the requirement that the location of the mutations must coincide with restriction endonuclease recognition sequences. To circumvent this limitation, primers that introduce a new restriction site have been employed in “primer-mediated” RFLP (Jacobson et al., “Rapid, Nonradioactive Screening for Activating Ras Oncogene Mutations Using PCR-Primer Introduced Restriction Analysis (PCR-PIRA),” PCR Methods Appl., 1(4):299 (1992); Chen et al., “A Method to Detect Ras Point Mutations in Small Subpopulations of Cells,” Anal. Biochem. 195(1):51–6 (1991); Di Giuseppe et al., “Detection of K-Ras Mutations in Mucinous Pancreatic Duct Hyperplasia from a Patient with a Family History of Pancreatic Carcinoma,” Am. J. Pathol., 144(5):889–95 (1994); Kahn et al., “Rapid and Sensitive Nonradioactive Detection of Mutant K-Ras Genes Via ‘Enriched’ PCR Amplification,” Oncogene, 6:1079–83 (1991); Levi et al., “Multiple K-Ras Codon 12 Mutations in Cholangiocarcinomas Demonstrated with a Sensitive Polymerase Chain Reaction Technique,” Cancer Research, 51(July):3497–502 (1991); and Mitsudomi et al., “Mutations of Ras Genes Distinguish a Subset of Non-Small-Cell Lung Cancer Cell Lines from Small-Cell Lung Cancer Cell Lines,” Oncogene, 6(8):1353–62 (1991)). However, subsequent investigators have demonstrated that errors are produced at the very next base by polymerase extension from primers having 3′ natural base mismatches (Hattori et al., “Mismatch PCR RFLP Detection of DRD2 Ser311Cys Polymorphism and Schizophrenia,” Biochem. Biophys. Res. Commun., 202(2):757–63 (1994); O'Dell et al., “PCR Induction of a TaqI Restriction Site at Any CpG Dinucleotide Using Two Mismatched Primers (CpG-PCR),” Genome Res., 6(6):558–68 (1996); and Hodanova et al., “Incorrect Assignment of N370S Mutation Status by Mismatched PCR/RFLP Method in Two Gaucher Patients,” J. Inherit. Metab. Dis., 20(4):611–2 (1997)). Such templates fail to cleave during restriction digestion and amplify as false-positives that are indistinguishable from true positive products extended from mutant templates.
Use of nucleotide analogs may reduce errors resulting from polymerase extension and improve base conversion fidelity. Nucleotide analogs that are designed to base-pair with more than one of the four natural bases are termed “convertides.” Base incorporation opposite different convertides has been tested (Hoops et al., “Template Directed Incorporation of Nucleotide Mixtures Using Azole-Nucleobase Analogs,” Nucleic Acids Res., 25(24):4866–71 (1997)). For each analog, PCR products were generated using Taq polymerase and primers containing an internal nucleotide analog. The products generated showed a characteristic distribution of the four bases incorporated opposite the analogs. Of significance, these products retained the original sequence at all natural base positions. Convertides readily form degenerate amplification products by virtue of their ability to assume different hydrogen bonding patterns through tautomeric shift (Brown et al., “Synthesis and Duplex Stability of Oligonucleotides Containing Adenine-Guanine Analogues,” Carbohydrate Research, 216:129–39 (1991)), bond rotation (Bergstrom et al., Nucleosides and Nucleotides, 15(1–3):59–68 (1996)), or base stacking (Bergstrom et al., Journal of the American Chemical Society, 117:1201–9 (1995); and Zhang et al., “Exploratory Studies on Azole Carboxamides as Nucleobase Analogs: Thermal Denaturation Studies on Oligodeoxyribonucleotide Duplexes Containing Pyrrole-3-Carboxamide,” Nucleic Acids Res., 26:2208–15 (1998)). Thus, PCR primers containing convertides may be used to facilitate base conversion. In principle, using the 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazine-7-one analog Q6), which is known to exhibit both the C-like and T-like tautomeric forms at the 3′ end of the primer (Brown et al., “Synthesis and Duplex Stability of Oligonucleotides Containing Adenine-Guanine Analogues,” Carbohydrate Research, 216:129–39 (1991)), a C-G base-pair may be converted to a T-A base pair (FIG. 1). Due to the better geometry, DNA polymerases may “read,” or extend better, from a Q6-G pair than a T-G mismatch (wobble base pair). Similarly, DNA polymerases may “write,” or incorporate both G and A bases opposite Q6 (Hill et al., “Polymerase Recognition of Synthetic Oligodeoxyribonucleotides Incorporating Degenerate Pyrimidine and Purine Bases,” Proc. Natl. Acad. Sci. USA, 95(8):4258–63 (1998)), whereas A is always inserted opposite a T base. Thus, the Q6 analog primer serves as an intermediary, providing a “preconversion” step before a natural base primer is added to selectively amplify the desired product from the degenerate pool. While nucleotide analogs have great potential, they have not been tested in high sensitivity assays. There is a need for a method that optimizes the fidelity of the analog conversion in the PCR process.
Optimization of PCR/RE/LDR
As discussed above, PCR used with a high fidelity conversion process would provide a valuable method for the amplification of mutant gene sequences. By designing primers with one or more mismatches, mutant DNA template can be efficiently extended, while poor extension is achieved on wild-type DNA template. However, once these primers extend with or without a mismatch, the products thereafter are perfect matches for the primer in subsequent PCR cycles. Thus, false positive signals are amplified in subsequent cycles. Moreover, PCR error can generate a base change in the template, which perfectly matches the primer. AS-PCR can detect pyrimidinepurine transversions at sensitivities of 1 in 105 (Newton et al., “Analysis of Any Point Mutation in DNA. The Amplification Refractory Mutation System (ARMS),” Nucleic Acids Res., 17(7):2503–16 (1989); and Tada et al., “Detection of Ras Gene Mutations in Pancreatic Juice and Peripheral Blood of Patients with Pancreatic Adenocarcinoma,” Cancer Res., 53(11):2472–4 (1993)). Nevertheless, the majority of cancer-associated mutations are CT and AG transitions, as, for example, are over 80% of p53 point mutations (de Fromentel et al., Genes Chromosomes Cancer, 4(1):1–15 (1992)). A DNA diagnostic method is needed to accurately quantify this type of low abundance mutation.
The ligation detection reaction (LDR) in conjunction with PCR has been used to quantify small amounts of PCR extension product. LDR uses two adjacent primers and a thermostable ligase to distinguish all four bases potentially found at any position in a DNA sequence (Barany, F., “Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase,” Proc. Natl. Acad. Sci. USA, 88:189–93 (1991); Barany, F., “The Ligase Chain Reaction in a PCR World,” PCR Methods Appl., 1:5–16 (1991); Day et al., “Detection of Steroid 21-Hydroxylase Alleles Using Gene-Specific PCR and a Multiplexed Ligation Detection Reaction,” Genomics, 29:152–62 (1995); and Khanna et al., Oncogene, 18:27–38 (1999)). Thermostable ligase demonstrates the highest fidelity when the discriminating base is located at the 3′ end of the upstream primer (Luo et al., “Improving the Fidelity of Thermus Thermophilus DNA Ligase,” Nucleic Acids Res., 24(15):3071–8 (1996)). PCR/LDR (PCR of a sequence from genomic DNA followed by LDR) can detect mutations with a sensitivity of approximately 1 mutant allele in 4,000 normal alleles (Khanna et al., Oncogene, 18:27–38 (1999)). Sensitivity of approximately 1 in 106 has been achieved by combining PCR with restriction endonuclease digestion of wild-type DNA (Sandy et al., Proc. Natl. Acad. Sci. USA, 89:890–4 (1992); and Pourzand et al., “Genotypic Mutation Analysis by RFLP/PCR,” Mutat. Res., 288(1):113–21 (1993)). Mutations occurring within the restriction site prevent cleavage of the mutant allele, while wild-type alleles bearing canonical restriction site sequence are depleted. As a result, subsequent PCR cycles preferentially amplify mutant DNA. If a mutation site is not within an endonuclease recognition site present in wild-type DNA, a restriction site must be introduced. This is typically done by PCR using a primer or primers with mismatched bases. Mutations cannot be detected in any portion of the restriction site spanned by the primers, since those bases are introduced directly through the primers. In a random DNA sequence, over 20% of bases are contained within a preexisting four-base restriction site and 60% of bases are within a four-base subsequence that can be converted into a restriction site by a single base change. In these small sites, 3′ terminal base mismatch primers must frequently be used. While conceptually straightforward, 3′ mismatch extension has proven to be difficult (Newton et al., “Analysis of Any Point Mutation in DNA. The Amplification Refractory Mutation System (ARMS),” Nucleic Acids Res., 17(7):2503–16 (1989); Kwok et al., “Effects of Primer-Template Mismatches on the Polymerase Chain Reaction: Human Immunodeficiency Virus Type 1 Model Studies,” Nucleic Acids Res, 18(4):999–1005 (1990); O'Dell et al., Genome Res. 6(6):558–68 (1996); and Day et al., Nucleic Acids Res., (1999)). The introduction of interrupted palindromic restriction sites has been more successful using internal mismatch primers spanning one half-site through the intervening bases up to the other half-site (Kumar et al., “Oncogene Detection at the Single Cell Level,” Oncogene 3(6):647–51 (1988); and Anderson et al., “Prevalence of RAS Oncogene Mutation in Head and Neck Carcinomas,” J. Otolaryngol., 21(5):321–6 (1992)). Several perfectly matched bases stabilize the 3′ end of the mismatch primer. However, this approach may be used only if the second half-site is present naturally in wild-type DNA. Mutations in the second half-site prevent digestion. Only mutations occurring at bases within the recognition sequence are detectable by RFLP methods. Mutations occurring at bases outside a preexisting restriction site in wild-type DNA may be detected by introducing a new restriction site containing that base.
Restriction endonucleases recognizing interrupted palindromes are less abundant than endonucleases recognizing contiguous four- and six-base sites. Multiple base changes would often be required to introduce an interrupted palindrome restriction site to identify mutations at any base.
More recently, methods of identifying known target sequences by probe ligation methods have been reported. U.S. Pat. No. 4,883,750 to N. M. Whiteley, et al.; D. Y. Wu, et al., Genomics 4:560 (1989), U. Landegren, et al., Science 241:1077 (1988), and E. Winn-Deen, et al., Clin. Chem. 37:1522 (1991). In one approach, known as oligonucleotide ligation assay (“OLA”), two probes or probe elements, that span a target region of interest, are hybridized to the target region. Where the probe elements base pair with adjacent target bases, the confronting ends of the probe elements can be joined by ligation, e.g., by treatment with ligase. The ligated probe element is then assayed, evidencing the presence of the target sequence.
In a modification of this approach, the ligated probe elements act as a template for a pair of complementary probe elements. With continued cycles of denaturation, hybridization, and ligation in the presence of pairs of probe elements, the target sequence is amplified linearly, allowing very small amounts of target sequence to be detected and/or amplified. This approach is referred to as ligase detection reaction. When two complementary pairs of probe elements are utilized, the process is referred to as the ligase chain reaction, which achieves exponential amplification of target sequences. F. Barany, “Genetic Disease Detection and DNA Amplification Using Cloned Thermostable Ligase,” Proc. Nat'l Acad. Sci. USA, 88:189–93 (1991) and F. Barany, “The Ligase Chain Reaction (LCR) in a PCR World,” PCR Methods and Applications, 1:5–16 (1991); Barany, U.S. patent application filed Mar. 19, 1999, Ser. No. 60/125,251. Techniques, such as PCR/LDR, that rely on mutant enrichment require optimization of reaction conditions in order to minimize random PCR errors. These errors would be indistinguishable from mutations originally present in clinical samples. One source of error-minimization may be found in optimization of the buffer conditions for PCR. Standard PCR buffers contain Tris, however the pKa of Tris is strongly dependent on temperature. A PCR reaction containing Tris pH 8.3 (measured at 23° C.) is approximately pH 7 near 65° C. (the extension temperature), and drops to approximately pH 6 near 95° C. (the template melting temperature). PCR error can result from template degradation and polymerase misincorporation. Template degradation occurs during periods of high temperature and low pH in each PCR cycle and limits product size in “long” PCR (Barnes, “PCR Amplification of up to 35-Kb DNA with High Fidelity and High Yield from Lambda Bacteriophage Templates,” Proc. Natl. Acad. Sci. USA, 91(6):2216–20 (1994); Cheng et al., “Effective Amplification of Long Targets from Cloned Inserts and Human Genomic DNA,” Proc. Natl. Acad. Sci. USA, 91(12):5695–9 (1994); and Sang et al., “Generation of Site-Directed Mutagenesis by Extralong, High-Fidelity Polymerase Chain Reaction,” Anal. Biochem., 233(1):142–4 (1996)). Raising the buffer pH in long PCR (using Tris 9.1) reduces the amount of template cleavage and increases PCR efficiency (Barnes, “PCR Amplification of up to 35-Kb DNA with High Fidelity and High Yield from Lambda Bacteriophage Templates,” Proc. Natl. Acad. Sci. USA, 91(6):2216–20 (1994)). Although the efficiency of long PCR increases with higher pH, the level of mutations within these PCR products may also increase since high pH decreases the fidelity of Taq and Pfu polymerases (Eckert et al., “High Fidelity DNA Synthesis by the Thermus Aquaticus DNA Polymerase,” Nucleic Acids Res., 18(13):3739–44 (1990); Eckert et al., “DNA Polymerase Fidelity and the Polymerase Chain Reaction,” PCR Methods Appl., 1(1):17–24 (1991); and Cline et al., “PCR Fidelity of Pfu DNA Polymerase and Other Thermostable DNA Polymerases,” Nucleic Acids Res., 24(18):3546–51 (1996)). Use of alternative PCR buffers with lower |ΔpKa| can improve polymerase fidelity and still reduce template damage by maintaining more neutral pH over a wider temperature range (Eckert et al., “DNA Polymerase Fidelity and the Polymerase Chain Reaction,” PCR Methods Appl., 1(1):17–24 (1991); and Brail et al., “Improved Polymerase Fidelity in PCR-SSCPA,” Mutat. Res., 303(4):171–5 (1993)). The addition of glycerol or formamide may reduce mutations arising from template damage during PCR cycling and may help avoid misextension from mispaired primers (Bottema et al., “PCR Amplification of Specific Alleles: Rapid Detection Of Known Mutations and Polymorphisms,” Mutat. Res., 288(1):93–102 (1993); and Cha et al., “Mismatch Amplification Mutation Assay (MAMA): Application to the C-H-Ras Gene,” PCR Methods Appl., 2(1):14–20 (1992)).
Thus, there is a need to improve buffer reaction conditions currently used in PCR, in order to minimize the opportunity for mismatches caused by PCR error. Increased analog conversion fidelity, alone, will not solve the need for an improved method of mutant DNA detection. In addition, there is a need to optimize PCR reaction conditions to decrease random PCR error, and finally, a method is needed that provides sensitive detection for the PCR extension products.